Background and purpose:Oxaliplatin (L-OHP) is one of the most commonly used chemotherapy drugs in colorectal cancer and L-OHP-resistance is very common in colorectal cancer therapy. This research was to discuss the reversal effect in L-OHP-resistant human colon cancer cell line LoVo/L-OHP by anemoside B4 (AB4) and tetrandrine (Tet) and to clarify their molecular mechanism. Methods:LoVo/L-OHP cells were treated for 48 h by AB4 and Tet at different concentrations to get non-toxic dose. Drug sensitivity was measured by MTT. After the treatment, the cell cycle and apoptosis of the cells were detected. Expression of P-gp mRNA, zDHHC9 mRNA and SMAD4 mRNA were detected by RT-PCR. Expression of P-gp, zDHHC9 and SMAD4 protein were detected by Western blot. Results:The IC50 of LoVo/L-OHP for L-OHP was (112.5±23.6) μg/mL, and the IC50 decreased to (62.8±21.4) μg/mL (P<0.05) and (58.9±26.3) μg/mL (P<0.05) after the treatment with 0.71 μg/mL AB4 and 0.45 μg/mL Tet (non-toxic dose) separately. The cell cycle experiment showed that the cells in G1 stage decreased and in S stage increased but with no statistical signiifcance (P>0.05). The cell apoptosis experiment showed that the apoptotic rate increased after the treatment with AB4 and Tet with non-toxic dose but with no statistical signiifcance (P>0.05). The RT-PCR experiment showed that the expressions of P-gp mRNA and zDHHC9 mRNA were increased and SMAD4 mRNA was decreased in LoVo/L-OHP cells compared with in LoVo cells (P<0.05), while it was found that P-gp mRNA in LoVo/L-OHP cells was decreased after the treatment with AB4 at non-toxic dose (P<0.05). Western blot experiment showed the protein of P-gp in LoVo/L-OHP cells was decreased after treatment with AB4 (P<0.05), which was accordant to the PCR result. The expression of zDHHC9 protein in LoVo/L-OHP cells was decreased in LoVo/L-OHP cells after treatment with Tet (P<0.05). Conclusion:AB4 and Tet have some reversal effect on resistant to L-OHP in LoVo/L-OHP cells. The molec-ular mechanism of the resistance reverse effect was related to down-regulation of P-gp for AB4 and down-regulation of zDHHC9 for Tet.

Objective To further elaborate the clinical characteristics,diagnosis,treatment and prognosis of primary cutaneous invasive epidermo-tropic CD8 positive cytotoxicity T-cell lymphoma.Methods The clinical characteristics,diagnosis and treatment of a patient with primary cutaneous invasive epidermotropic CD8 positive cytotoxicity T-cell lymphoma were summarized,and diagnosis and treatment of the disease from published literature were reviewed.Results After one year long,recurrence of exudative maculopapule on his body and limbs,a middle-aged male patient visited our hospital.Then,the patient was diagnosed as T2N0M0 Ⅰ phase of primary cutaneous invasive epidermo-tropic CD8 positive cytotoxicity T-cell lymphoma based on results of skin biopsy,immunohistochemistry,T-cell gene rearrangement and PET-CT.Through integrative medicine treatment (combination of bruceolic oil emulsion and chemotherapy with GDP regimen),the patient achieved complete remission (CR).The treatment and follow-up continues till now and his condition was stable.Conclusions Clinical occurrence of primary cutaneous invasive epidermo-tropic CD8 positive cytotoxicity T-cell lymphoma is rare and early diagnosis is difficult.It could be diagnosed with histopathology combined with T-cell gene rearrangement.In order to avoid misdiagnosis,great caution should be taken during disease history inquiry and physical examination,should be early lesion for delayed healing of recurrent lesions,biopsy should be conducted as soon as possible and immunohistochemistry should be performed simultaneously,which will provide a good basis for further treatment.

Objective To develop a simple method of determination of sorafenib in serum by reversed-phase high performance liquid chromatography (RP-HPLC) and to explore its application in sorafenib therapeutic drug monitoring (TDM).Methods Sorafenib extracted by ethyl ether-petroleum (9∶1) with internal standard of erlotinib from serum was wiped off in 60 ℃ water bath.Sorafenib was redissolved by mobile buffer and analyzed by 40 μl.Chromatographic column was Symmetry Rp18 (5 μm,4.6 mm×250 mm,waters) column in normal temperature.The mobile buffer was 28 mmol/L acetate buffer (pH 5.8)-acetonitrile (37∶63).Sorafenib and erlotinib were detected in 249 nm and 335 nm,respectively.Results The concentration range of sorafenib was 0.50-20.00 μg/ml (r =0.9999).The within-day and between-day accuracies of sorafenib were less than 4.77 ％ and 8.79 ％,respectively.The average recovery rate was 98.48 ％.Sorafenib was stable in serum or after extraction.The concentrations of sorafenib in two patients were detected.Conclusion Detection of sorafenib in serum by RP-HPLC is simple and accurate,which is available to determine sorafenib in serum.The TDM of sorafenib has clinical significance.

Objective To investigate the imaging features in CT/MR of pancreatic neuroendocrine tumors(PNETs) with multiple lesions and further deepen the understanding of this disease .Methods A retrospective review of 12 PNETs patients′radiological data with pancreatic tumors′numbers≥2 and confirmed by surgery or fine needle aspiration biopsy in Changhai Hospital were conducted .Five cases underwent pancreatic CT plain and enhanced scan , 2 cases underwent MRI plain and enhanced scan , and 5 cases underwent both CT and MRI scan .Results There were totally 46 lesions in 12 patients.There were 29 (63.0%) lesions located in the pancreatic head and neck , and 17(37.0%) lesions located in body and tail of pancreas.The sizes of the lesions ranged from 0.8 to 9.5 cm,and the median size was 2.9 cm.Forty-four (95.7%) of the tumors was round or oval , and 2 ( 4.3%) was lobulated;44 ( 95.7%) mass solid and 2 (4.3%) was cystic.CT plain scan detected punctate , crescent or nodular calcification in 8(17.4%) lesions;enhanced scan found 42 lesions(91.4%) were markedly enhanced in the arterial phase , 2 lesions (4.3%) were markedly enhanced in the pancreatic phase;2 lesions (4.3%) were slightly enhanced and the degree of enhancement was lower than that of the normal pancreas .Four cases (33.3%) had dilatation of pancreatic duct and/or the bile duct, 4 cases (33.3%) had distant organ metastasis, 2 cases (16.7%) had lymph node metastasis, and 3 cases (25.0%) had vascular invasion .Conclusions PNETs can be multiple and vary in the size.Most of the lesions are round or oval solid lesions and the malignant signs for organ metastasis can be found occasionally .In dynamic enhanced scanning , the obvious enhancement of the solid portion in the tumor and the higher enhancement degree than that of normal pancreas is the main characteristic .

Objective To screen serum biomarkers in patients with hepatocelhlar carcinoma by using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry(SELDI-TOF-MS)technique and to evaluate its clinical implication in the patients whose alpha-fetoprotein were negative in the sera. Methods Proteomic spectra were generated by mass spectroscopy in 112 cases, including 57 cases AFP-negative hepatocellular carcinoma,and 55 cases of healthy control. The consequence was analyzed and the characteristic preteomic peaks was selected by using Biomarker Wizard. Results Seven low expressed potential biomarker were indentified with the mass-to-charge ratio of 4.2×103, 4.1×103, 6.7×103, 5.7×103, 6.5×103, 6.9×103, 5.8×103. The sensitivity for diagnosing hepatic cancer were 88.23 % and specificity was 92.31%. These peaks were not correlated with age, sex, tumor mass size, pathology grading and cirrhosis in hepatocellular carcinoma. Conclusion SELDI-TOF-MS offers a unique platform for the proteomic detection of hepatocellular carcinoma.It also offers an auxiliary diagnosis method to the patients whose alpha-fetoprotein are negative in the serum.

Objective:To investigate the value of machine learning model based on MRI in predicting the abundance of tumor infiltrating CD 8+ T cell and prognosis of pancreatic cancer patients. Methods:The clinical data of 156 patients with pathological confirmed pancreatic cancer who underwent pre-operative MRI within 7 days before surgery in the First Affiliated Hospital of Naval Medical University from January 2017 to April 2018 was retrospectively analyzed. According to the international consensus on the predictive model, a total of 116 patients from January to December 2017 were included in the training set, and a total of 40 patients from January to April 2018 were included in the validation set. With the overall survival of patients as the outcome variable, X-Tile software was used to obtain cut-off values of the percentage of CD 8+ T cells, and all patients were divided into CD 8+ T-high and -low groups. The clinical, pathological and radiological features were compared between two groups. 3D slicer software was used to draw the region of interest in each layer of the primary MR T 1- and T 2-weighted imaging, arterial phase, portal venous phase, and delayed phase images for tumor segmentation. Python package was applied to extract the radiomics features of pancreatic tumors after segmentation and the extracted features were reduced and chosen using the least absolute shrinkage and selection operator (Lasso) logistic regression algorithm. Lasso logistic regression formula was applied to calculate the rad-score. The extreme gradient boosting (XGBoost) were used to construct the machine learning predicted model. The models′ performances were determined by area under the ROC curve (AUC), sensitivity, specificity, accuracy, positive predictive value, and negative predictive value. Results:The cut-off value of the CD 8+ T-cell level was 19.09% as determined by the X-tile program. Patients in the high CD 8+ T cell group had a longer median survival than those in the low CD 8+ T cell group (25.51 month vs 22.92 month, P=0.007). The T stage in the training set and tumor size in the validation set significantly differed between the groups (all P value <0.05). A total of 1 409 radiomics features were obtained, and 19-selected features associated with the level of CD 8+ T cell were determined after being reduced by the Lasso logistic regression algorithm. The rad-score was significantly lower in the CD 8- high group (median: -0.43; range: -1.55 to 0.65) than the CD 8- low group (median: 0.22; range: -0.68 to 2.54, P<0.001). The prediction model combined the radiomics features and tumor size. In the training set, the AUC, sensitivity, specificity, accuracy, and positive and negative predictive value were 0.90 (95% CI 0.85-0.95), 75.47%, 90.48%, 0.84, 0.87, and 0.81. In the validation set, the AUC, sensitivity, specificity, accuracy, and positive and negative predictive value were 0.79 (95% CI 0.63-0.96), 90.00%, 80.00%, 0.85, 0.82, and 0.89. The predictive model can accurately distinguish patients with high and low CD 8+ T cells in pancreatic cancer. Conclusions:The radiomics-based machine learning model is valuable in predicting the CD 8+ T cells infiltrating level in pancreatic cancer patients, which could be useful in identifying potential patients who can benefit from immunotherapies.

Objective:To identify the disease-causing mutation in a Chinese family with Stickler syndrome type 1.Methods:The pedigree investigation was conducted.A Chinese family with Stickler syndrome type 1 was enrolled in the Shantou International Eye Center in June 2012.Medical history collection and clinical examinations, such as vision, intraocular pressure, slit lamp microscopy and fundus, were carried out in all the included family members and the diagnosis was made by clinical experts.Total genomic DNAs were extracted from the peripheral blood samples (5 ml) obtained from 5 patients and 4 healthy members.The potential variant of the proband's father Ⅲ-5 were screened by whole exome sequencing (WES) and stepwise bioinformatic analysis.The segregation and mutation conformation of the variant was verified by Sanger sequencing.The pathogenicity of the variant was predicted by SIFT, Polyphen2, and MutationTaster.Conservation and three-dimensional structure of amino acid mutation were analyzed by multiple sequence alignment and UniProt.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Joint Shantou International Eye Center (No.EC20110310[2]-P02).Written informed consent was obtained from each subject or the guardian.Results:An autosomal dominant inherence in 39 members of 4 generations including 15 patients and 24 phenotypically normal members was found in the family.The proband (Ⅳ-4) showed high myopia, retinal detachment and strabismus in the right eye, and the left eye was blind.A patient (Ⅲ-5) showed high myopia and cataract in the right eye, atrophy in the left eye.A patient (Ⅳ-9) showed binocular high myopia.A heterozygous variation, c.1693C>T: p.Arg565Cys, within the exon 26 of COL2A1 gene was revealed in patient Ⅲ-5, which was only found in the patients and not in phenotypically normal members, indiacating co-separation in this family.The variant was predicted to be a severe damage by SIFT, Polyphen2 and MutationTaster.The amino acid mutation at position 565 was highly conservative among human, mouse, rat, bovine and Xenopus laevis, which caused the arginine to cysteine substitution at the X position in triple helix repeat region Gly-X-Y, affecting the function of fibrous protein and becoming pathogenic. Conclusions:Variant c.1693C>T: p.Arg565Cys in COL2A1 gene is disease-causing in this family and this is the first report about the variant in China.

Objective: To investigate the protective effect of propofol on neurological function in rats after traumatic brain injury (TBI) and its possible mechanism. Methods: A total of 96 SD rats were randomly divided into sham operation group, sham operation+ propofol group, TBI group and TBI + propofol group, with 24 rats in each group. The TBI model was prepared by modified Feeney method. The sham operation+ propofol group and the TBI+ propofol group were given 50 mg/kg of propofol once daily. The sham operation group and the TBI group were injected with the same amount of normal saline. Modified neurobehavioral functional scores (mNSS) were evaluated at 1, 3, 7 and 14 days after injury; dry-wet specific gravity method was used to detect brain water content in injured area; TUNEL staining was used to detect neuronal apoptosis; chemiluminescence was used to detect activity of Oxygen cluster (ROS) content; Western blot was used to determine the expressions of inositol requirement enzyme 1 (IRE-1), enhancer binding protein homolog protein (CHOP), heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and nuclear factor E2 related factor 2 (Nrf2) protein. Results: Compared with the sham operation group and the sham operation + propofol group, the mNSS, brain tissue water content, apoptosis number and ROS increased at 1, 3, 7 and 14 days after TBI in the TBI group and TBI + propofol group (P<0.05). Compared with TBI group, mNSS in TBI+ propofol group decreased significantly [(9.3±1.4)points ∶(10.9±1.2)points] 7 days after injury (P<0.05); the brain tissue water content decreased significantly [(81.0±0.8)%∶(82.1±0.8)%] 3 days after injury (P<0.05); the number of apoptotic cells decreased significantly 7 days after injury[(14.1±1.4)%∶(15.6±1.6)%], with the most significant decrease at 14 days after injury [( 10.4±1.5)%∶(13.2±1.4)% (P<0.05); and ROS decreased significantly 7 days after injury [(61.5±4.0)RFU∶(77.3±5.5)RFU](P<0.05). Compared with the sham operation group and the sham operation+ propofol group, the expressions of IRE-1 and CHOP were significantly up-regulated in the TBI group and the TBI+ propofol group (P<0.05); the expressions of HO-1, NQO1 and Nrf2 in the TBI group were significantly decreased (P<0.05); the expressions of HO-1 and NQO1 in TBI+ propofol group were increased (P<0.05) while the expression of Nrf2 were decreased slightly (P<0.05). Compared with the TBI group, the expressions of IRE-1 and CHOP in TBI+ propofol group were decreased (P<0.05), while the expressions of HO-1, NQO1 and Nrf2 were significantly increased (P<0.05). Conclusion After TBI in rats, propofol can reduce oxidative stress by activating the Nrf2-antioxidant element (ARE) pathway, reduce brain edema, and inhibit neuronal apoptosis, thus playing a neuro-protective role.

OBJECTIVE：To establish the method for the content determination of 7 components，such as puerarin ， 3′-methoxypuerarin，daidzein，rutin，hesperidin，salvianolic acid A and quercetin ，in Zhengxin jiangzhi tablets ，and conduct cluster heatmap analysis. METHODS ：HPLC method was adopted. The separation was performed on Kromasil C 18 column with mobile phase consisted of acetonitrile- 0.1％ formic acid solution （gradient elution ）at the flow rate of 0.8 mL/min. The detection wavelength was set at 280 nm，and the column temperature was 25 ℃. The sample size was 10 μL. Taking the content data as the object，the cluster heatmap was drawn by Hiplot scientific research mapping platform. RESULTS ：The linear range of puerarin ， 3′-methoxypuerarin，daidzein，rutin，hesperidin，salvianolic acid A and quercetin were 17.00-170.00（r＝0.999 9），5.14-51.40（r＝ 0.999 8），3.00-30.00（r＝0.999 8），153.00-1 530.00（r＝0.999 9），7.88-78.75（r＝0.999 8），2.85-28.50（r＝0.999 9）and 11.34-113.40 μg/mL（r＝0.999 8），respectively. RSDs of precision ，stability（24 h）and repeatability tests were all less than 2％； the average recoveries were 99.58％（RSD＝0.83％，n＝6），100.31％（RSD＝1.17％，n＝6），100.61％（RSD＝1.08％，n＝6）， 100.05％（RSD＝0.82％，n＝6），100.31％（RSD＝1.38％，n＝6），100.31％（RSD＝0.85％，n＝6），99.85％（RSD＝1.01％， n＝6），respectively. The contents of above components in 10 batches of samples were 7.262 5-8.941 5，2.464 9-3.068 9，1.478 9- 1.883 4，58.632 8-79.408 3，3.569 4-4.500 6，1.077 6-1.341 5，1.139 7-5.957 0 mg/g，respectively. Results of cluster heatmap analysis showed that 10 batches of samples could be divided into 4 categories，including S 1-S3 as one category ，S4 as one category，S5-S6 as one category and S 7-S10 as one category. CONCLUSIONS ：The established method is simple ，accurate and specific，which can be used for the quality control of Zhengxin jiangzhi tablets ，combined with cluster heatmap analysis. There are some differences in the quality of different batches of samples.

Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered. Here, we report that cardiomyocytes (CMs) experience epigenetic and transcriptional decline of cardiac gene expression immediately after birth, leading to a transition state of CMs at postnatal day 7 (P7) that was essential for CM subtype specification during heart maturation. Large-scale single-cell analysis and genetic lineage tracing confirm the presence of transition state CMs at P7 bridging immature state and mature states. Silencing of key transcription factor JUN in P1-hearts significantly repressed CM transition, resulting in perturbed CM subtype proportions and reduced cardiac function in mature hearts. In addition, transplantation of P7-CMs into infarcted hearts exhibited cardiac repair potential superior to P1-CMs. Collectively, our data uncover CM state transition as a key event in postnatal heart maturation, which not only provides insights into molecular foundations of heart maturation, but also opens an avenue for manipulation of cardiomyocyte fate in disease and regenerative medicine.
Gene Expression Regulation ; Heart ; Myocytes, Cardiac/metabolism* ; Single-Cell Analysis ; Transcription Factors/metabolism*